Method of treating milk fever

ABSTRACT

The present invention relates to a method of preventing milk fever which comprises administering to a preparturient female ruminant an effective amount of 24,24-difluoro-1α,25-dihydroxycholecalciferol.

BACKGROUND OF THE INVENTION

The invention relates to a method for the prevention of milk fever inpreparturient female ruminants which comprises administering to apreparturient female ruminant an effective amount of24,24-difluoro-1α,25-dihydroxycholecalciferol.

Vitamin D₃ or cholecalciferol is a well-known agent for the control ofcalcium and phosphorous homeostasis. In the normal animal or human, thiscompound is known to stimulate intestinal calcium transport andbone-calcium mobilization and is effective in preventing rickets.

It is also now well known that to be effective, vitamin D₃ must beconverted in vivo to its hydroxylated forms. For example, the vitamin isfirst hydroxylated in the liver to form 25-hydroxy-vitamin D₃ and isfurther hydroxylated in the kidney to produce 1α,25-dihydroxy vitamin D₃or 24R,25-dihydroxy vitamin D₃. The 1α,25-dihydroxylated form of thevitamin is generally considered to be the physiologically-active orhormonal form of the vitamin and to be responsible for what are termedthe vitamin D-like activities such as increasing intestinal absorptionof calcium and phosphate and mobilizing bone mineral.

SUMMARY OF THE INVENTION

The present invention relates to a method for the prevention of milkfever in female preparturient ruminants which comprises administering toa female preparturient ruminant an effective amount of24,24-difluoro-1α,25-dihydroxycholecalciferol.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification and the appended claims, the term"lower alkyl" denotes a monovalent substituent consisting solely ofcarbon and hydrogen of from 1 to 8 carbon atoms which may be straight-or branched-chain. Examples of lower alkyl groups are methyl, ethyl,n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and the like. Theterm "lower alkylene" denotes a divalent substituent consisting solelyof carbon and hydrogen of from 1 to 8 carbon atoms which may bestraight- or branched chain and whose free valences are attached to twodistinct groups. Examples of alkylene groups are methylene, ethylene,propylene, butylene, amylene, hexylene, heptylene, octylene and thelike. The term "lower alkoxy" refers to a lower alkyl ether group.Examples of alkoxy groups are methoxy, ethoxy, isopropoxy, tert-butoxyand the like. The term "phenyl-lower alkoxy" refers to a lower alkoxygroup which is substituted by a phenyl ring. Examples of phenyl-loweralkoxy groups are benzyloxy, 2-phenylethoxy, 4-phenylbutoxy and thelike. The term "alkanoyloxy group" refers to the residue of an aliphaticcarboxylic acid of from 1 to 8 carbon atoms formed by removal of thehydrogen from the hydroxyl portion of the carboxyl group. Examples ofalkanoyloxy groups are formyloxy, acetoxy, butyroyloxy, hexanoyloxy andthe like. The term "substituted" as applied to "phenyl" refers to phenylwhich is substituted with one or more of the following groups: alkyl,halogen (that is, fluorine, chlorine, bromine or iodine), nitro, cyano,trifluoromethyl and the like. The term aralkyl denotes a radical inwhich a lower alkyl H atom is substituted by an aryl group. Exemplary ofaralkyl are benzyl, phenylethyl, phenylpropyl and the like. The termaryl denotes an organic radical derived from an aromatic hydrocarbon bythe removal of a hydrogen atom. Exemplary of aryl are phenyl andsubstituted phenyl. The term alkanoyl denotes the residue of analiphatic carboxylic acid of from 1 to 8 carbon atoms formed by theremoval of hydroxyl from the carboxyl group. Exemplary of alkanoyl areacetyl, propionyl, butyroyl, pentanoyl and the like. The term aralkanoyldenotes an alkanoyl radical in which one hydrogen of the alkyl portionof the molecule has been substituted by aryl. Exemplary of aralkanoylare phenylacetyl, phenylpropionyl, phenylbutyroyl, phenylpentanoyl andthe like. The term aroyl denotes the residue of an aromatic carboxylacid of from 7 to 20 carbon atoms formed by the removal of a hydroxygroup from the carboxyl group. Exemplary of aroyl are benzoyl, toloyland the like. The term acyl denotes the residue of an aliphatic oraromatic carboxylic acid formed by the removal of the hydroxyl portionof the carboxyl group. Exemplary of acyl are aroyl and alkanoyl.

In the steroid formulas presented herein, the various asymmetricsubstituents are illustrated as joined to the steroid nucleus by one ofthese notations: a solid line ( ) indicating a substituent which is inthe β-orientation, that is, above the plane of the molecule, a dottedline ( ) indicating a substituent which is in the α-orientation, thatis, below the plane of the molecule or a wavy line ( ) indicating asubstituent which may be in the α- or β-orientation. The formulas haveall been drawn to show the compounds in their absolute stereochemicalconfigurations. Since the starting materials are derived from anaturally-occurring steroid (3β-hydroxyandrost-5-en-17-one) the productsexist in the single absolute configuration depicted herein. However, theprocesses of the present invention are intended to apply as well to thesynthesis of steroids of the "unnatural enantiomer" and racemic series,that is, the epimers of the compounds depicted herein and mixtures ofboth. Thus, one may begin the synthesis utilizing "unnatural" or racemicstarting materials to prepare either "unnatural" or racemic products,respectively.

The Greek letter xi (ξ) in the name of a vitamin D₃ intermediate ormetabolite indicates that the stereochemistry of the substituent towhich it refers is undefined or that the product consists of a mixtureof compounds epimeric at the designated position.

The nomenclature adopted to define absolute configuration ofsubstituents bound to carbon atom 20 of the steroid nucleus is describedin the Journal of Organic Chemistry, 34 (1970) 2849 under the title"IUPAC Tentative Rules for the Nomenclature of Organic Chemistry.Section E. Fundamental Stereochemistry".

In the process for the preparation of24,24-difluoro-1α,25-dihydroxycholecalciferol of the formula ##STR1##wherein F is fluorine, is prepared by treating a compound of the formula##STR2## wherein F is fluorine and

R, R¹, and R², independently, are lower alkyl, aralkyl, aryl, tri-loweralkylsilyl, di-lower alkylarylsilyl, lower-alkyldiarylsilyl,triarylsilyl or a group of the formula ##STR3## wherein R³ is hydrogenor lower alkyl;

R⁴ and R⁵ each independently are lower alkyl and R⁴ and R⁵ takentogether are lower alkylene of from 3 to 6 carbon atoms,

with an acid or tri-lower alkylsilyl iodide, or is prepared by treatinga compound of the formula ##STR4## wherein F is as above and

R', R^(1') and R^(2') are independently, hydrogen, lower alkanoyl,aralkanoyl or aroyl, with a saponifying agent.

The starting materials for the invention are pregn-5-en-21-oic acidesters of the formula ##STR5## wherein R and R¹ are as above and

R⁶ is lower alkyl, aralkyl, or aryl.

The compound of formula V can be prepared according to the followingmethodology starting with the known 1α,3β-dihydroxyandrost-5-en-17-one(Formula V-A) [R. M. Dodson, A. H. Goldkamp and R. D. Muir, J. Amer.Chem. Soc., 82 4026 (1960)]. The sequence (formula V-A→formula V)parallels the work of J. Wicha and K. Bal, J. Chem. Soc., Perkin Trans I(1978) 1282.

An intermediate of formula V, which is utilized hereinafter, can beprepared as follows: ##STR6## wherein R, R¹ and R⁶ are as above.

The hydroxy groups in [1α,3β]-1,3-dihydroxyandrost-5-ene-17-one (formulaV-A) were protected as acetals, ethers, or silyl ethers to yieldcompounds of of formula V-B, as described below.

To form acetal protecting groups the compound of formula V-A was treatedwith lower alkyl or aromatic vinyl ethers and a catalytic amount of astrong acid such as p-toluenesulfonic acid or hydrochloric acid in aninert solvent such as the ethers, diethyl ether or tetrahydrofuran orinert organic solvents such as benzene, toluene, or methylene chlorideat a temperaure range of -50° C. to 50° C., most preferably -50° C. to0° C. to yield acetal compounds of formula V-B.

Exemplary of lower alkyl vinyl ethers are methyl vinyl ether, ethylvinyl ether, methyl 1-methylvinyl ether, methyl 2-methylvinyl ether andthe like. Exemplary of aromatic ethers are phenyl vinyl ether, phenyl1-methyl-vinyl ether, phenyl 2-methyl vinyl ether and the like. Theaforementioned aromatic ethers may be substituted, and are exemplifiedby 4-methylphenyl vinyl ether, 4-chlorophenyl vinyl ether,4-methylphenyl 1-methylvinyl ether and the like. The aforementionedlower alkyl vinyl ethers may include cyclic vinyl ethers(3,4-dihydro-2H-pyrans and 2,3-dihydro-2H-furans).

To form ether protecting groups the compound of formula V-A was treatedwith lower alkyl, aralkyl or aryl halide and a tertiary amine oraromatic tertiary amine base in an inert solvent such as benzene,toluene or methylene chloride, an ether solvent such as diethyl ether ortetrahydrofuran or a tertiary amine solvent such as tiethylamine orpyridine. Suitable tertiary amine or aromatic tertiary amine basesinclude triethyl amine, pyridine, s-collidine and4-dimethylaminopyridine. Suitable lower alkyl halides include methyliodide, ethyl iodide and the like. Suitable aralkyl halides includebenzyl chloride, p-methoxybenzyl chloride, p-nitrobenzyl bromide and thelike. Suitable aryl halides include iodobenzene, p-nitrophenyl iodideand the like. The reactions are typically carried out at a temperaturerange of -20° C. to 100° C. to yield ether compounds of formula V-B.

To form silyl ether protecting groups the compound of formula V-A wastreated with tri-lower alkylsilyl halides such as trimethylsilylchloride, trimethylsilyl bromide, and t-butyl dimethylsilyl chloride,di-lower alkylarylsilyl halides, such as dimethylphenylsilyl chloride,lower alkyldiarylsilyl halides such as methyldiphenylsilyl chloride, andtriarylsilyl halides such as triphenylsilyl iodide and a tertiary aminebase such as triethylamine or imidazole or an aromatic tertiary aminebase such as pyridine or 4-dimethylaminopyridine. Suitable solventsinclude polar aprotic solvents such as dimethylformamide, inert solventssuch as benzene, toluene, and methylene chloride, and ether solventssuch as diethyl ether and tetrahydrofuran. Suitable temperatures include-20° C. to 100° C., most preferably 0° C. to 50° C. to yield silyl ethercompounds of formula V-B.

The compounds of formula V-B were treated with a Horner-Wittig reagentand a base to yield compounds of the formula V-C. Suitable Horner-Wittigreagents include lower alkylphosphonoacetates such astriethylphosphonoacetate, aralkyl phosphonoacetates such as benzyldiethylphosphonoacetate and arylphophosphonoacetates such as phenyldiethylphosphonoacetate and triphenylphosphonoacetate. Suitable basesand solvents include alkali metal alkoxides such as sodium ethoxide inethanol, potassium methoxide in methanol and the like and alkali metalhydrides such as sodium hydride and potassium hydride in inert solventssuch as the ethers, diethyl ether, tetrahydrofuran and the like or polaraprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide.Suitable temperatures are in the range of from -20° C. to 120° C., mostpreferably at 20° C. to 80° C.

A compound of formula V-C is converted to a compound of formula V bycatalytic hydrogenation over a noble metal catalyst. Suitable noblemetal catalysts include platinum oxide, platinum metal, platinum oncharcoal, palladium metal, and palladium on charcoal. Suitable solventsinclude lower alkanols of 1 to 8 carbon atoms such as methanol andethanol, carboxylic acid esters such as ethyl acetate, inert solventssuch as methylene chloride, and ether solvents such as tetrahydrofuran,diethyl ether, p-dioxane and the like. The reactions are typically rununder 1 atmosphere of hydrogen at temperatures of 0° C. to 50° C., mostpreferably at 0°-30° C.

In the process described herein, a pregn-5-en-21-oic acid ester of theformula ##STR7## wherein R, R¹ and R⁶ are as above, is treated with anorganometallic reagent to yield as metallated pregn-5-en-21-oic acidester of the formula ##STR8## wherein R, R¹ and R⁶ are as above and

M is lithium, sodium, potassium, magnesium/2 or zinc/2.

For example, the lithium organometallic compound VI may be formed byreaction of the compound of formula V with, for example, lithiumdiisopropylamide. The sodium organometallic compound VI may be formed byreaction of the compound of formula V with, for example, sodiumhexamethyl-disilazane. The potassium organometallic compound VI may beformed by reaction with the compound of formula V with, for example,potassium hydride.

The metallated pregn-5-en-21-oic acid ester of formula VI is preferablygenerated in situ and is then reacted with a compound of the formula##STR9## wherein X is iodo, bromo, chloro, lower alkyl sulfonyloxy,phenylsulfonyloxy or substituted phenylsulfonyloxy;

F is fluorine and

R² is as above,

to yield the alkylated compound of the formula ##STR10## wherein F, R,R¹ and R⁶ are as above.

The aforementioned reaction sequence (V→VI+VII→VIII) starting with thecompound of formula V may be carried out in aprotic inert solvents suchas, for example, ethers, for example, diethyl ether, tetrahydrofuran,dimethoxyethane, dioxane and the like; amides, for example,hexamethyl-phosphoramide and the like. Preferred solvents for thispurpose are tetrahydrofuran and hexamethylphosphoramide. The use oftetrahydrofuran-hexamethylphosphoramide mixtures is particularlypreferred.

The alkylation reaction between a compound of formula VI and formula VIIis conveniently carried out at a temperature between -78° C. and 60° C.Most preferably, the alkylation reaction is conducted between atemperature of about -40° C. to 0° C. The desired alkylation product offormula VIII, containing the desired 20R-absolute configuration, can beisolated by the usual chemical and physical means such as chromatographyand in this manner can be separated from any undesired impurities suchas materials of formula V and VII.

The preparation of a compound of formula VII, which is utilizedhereinafter, can be prepared as follows: ##STR11## wherein F, R², and R⁶are as above and

R⁷ is lower alkanoyl, aralkanoyl, and aroyl.

The commercially available 2,2-difluorosuccinic acid of formula VII-A istreated with a 1:1 mixture of an acid anhydride and an organic acidchloride at temperatures in the range of 0° C. to reflux, preferably 0°C. to room temerature so as to yield a cyclic anhydride of the formulaVII-B.

The cyclic anhydride of formula VII-B is selectively opened to themonoester of formula VII-C by reaction with an excess of a loweraliphatic alcohol such as methanol, ethanol, propanol, isopropanol,butanol, isobutanol or an araliphatic alcohol such as benzyl alcohol, oran aromatic alcohol such as phenol and the like. The reaction is carriedout at temperatures in the range of 0° C. to reflux preferably 0° C. to50° C. to yield a compound of the formula VII-C.

The monester of formula VII-C is selectively reduced with diborane in alower aliphatic ether solvent such as diethyl ether or ether solventsuch as tetrahydrofuran, or borane-dimethyl sulfide complex in a loweraliphatic organic solvent such as hexane and methylene chloride, orinert aromatic solvent such as benzene, toluene and the like, so as toyield the hydroxy ester of formula VII-D. The foregoing reaction iscarried out at temperatures in the range of -20° C. to room temperatureto yield a compound of the formula VII-D.

The hydroxy ester of formula VII-D is converted to the diol of theformula VII-E by treatment with excess methyl organometallic reagentsuch as methyllithium, methylmagnesium chloride, methylmagnesiumbromide, methylmagnesium iodide and dimethylmagnesium in ether solventsuch as diethyl ether and tetrahydrofuran. The foregoing reaction may becarried out at temperatures in the range of -20° C. to 50° C.,preferably -10° C. to room temperature.

The diol of formula VII-E is selectively protected by treatment with anacylating agent such as acetic anhydride or benzoyl chloride and anaromatic tertiary amine such as pyridine so as to yield the compound ofthe formula VII-F.

The tertiary alcohol group of formula VII-F is then protected usinglower alkyl or aromatic vinyl ethers and p-toluenesulfonic acid catalystin inert ethers such as diethyl ether or other inert organic solventsuch as lower aliphatic hydrocarbons so as to yield an acetal of theformula VII-G.

Exemplary of lower alkyl vinyl ethers are methyl vinyl ether, ethylvinyl ether, methyl 1-methylvinyl ether, methyl 2-methylvinyl ether andthe like. Exemplary of aromatic ethers are phenyl vinyl ether, phenyl1-methyl-vinyl ether, phenyl 2-methyl vinyl ether and the like. Theaforementioned aromatic ethers may be substituted, and are exemplifiedby 4-methylphenyl vinyl ether, 4-chlorophenyl vinyl ether,4-methylphenyl 1-methylvinyl ether and the like. The aforementionedlower alkyl vinyl ethers may include cyclic vinyl ethers(3,4-dihydro-2H-pyrans and 2,3-dihydro-2H-furans).

The ester group of formula VII-G is removed by reduction with lithiumaluminum hydride in ether solvents such as diethyl ether ortetrahydrofuran at temperatures in the range of -20° C. to refluxpreferably -10° C. to room temperature. This transformation could alsobe accomplished by a saponification reaction with alkali metalhydroxides such as potassium hydroxide or alkali metal alkoxides such assodium methoxide in lower aliphatic alcohol or lower aliphaticalcohol-water solvent systems at a temperature range of -20° C. to roomtemperature to yield an acetal alcohol of the formula VII-H.

The acetal alcohol of formula VII-H is then converted to the acetalester of the formula VII-I, with a sulfonyl halide such asp-toluenesulfonyl chloride and an aromatic amine such as pyridine. Othersulfonyl halides such as lower alkyl sulfonyl halides exemplified bymethanesulfonyl chloride or aromatic sulfonyl halides exemplified bybenzenesulfonyl bromide and p-nitrobenzenesulfonyl chloride may also beused at a temperature range of -20° C. to 50° C., most preferably at-10° C. to 30° C.

Displacement of the organic sulfonate group in formula VII-I isaccomplished by reaction with an alkali metal halide such as sodiumiodide in a lower alkyl ketone such as acetone so as to yield the acetalhalide of the formula VII-J.

The above reaction is preferably carried out with the addition of anacid scavenger such as an hindered tertiary amine base which isexemplified by diisopropyl ethyl amine. This reaction is carried out attemperatures in the range of -20° C. to 50° C., preferably at -10° C. toroom temperature. The compounds of formula VII-I and VII-J areencompassed by general formula VII.

Thereafter, the compound of formula VIII is reduced to a compound of theformula ##STR12## wherein F, R, R¹ and R² are as above, by reduction ofthe R⁶ ester grouping of formula VIII with a complex metal hydridereducing agent. Suitable complex metal hydride reducing agents for thispurpose include alkali metal aluminum hydrides such as lithium aluminumhydride; mono-, di- or tri-(lower alkoxy) alkali metal aluminum hydridessuch as, for example, lithium tris(tert-butoxy) aluminum hydride; mono-,di- or tri(lower alkoxy lower alkoxy) alkali metal aluminum hydridessuch as, for example, sodium bis(2-methoxyethoxy) aluminum hydride;di(lower alkyl) aluminum hydrides such as, for example, diisobutylaluminum hydride; and so forth. A particularly-preferred complex metalreducing agent for this purpose is lithium aluminum hydride. Suitablesolvents for this reduction include ethers such as diethyl ether,tetrahydrofuran, dimethoxyethane and dioxane. The reduction isconveniently carried out at a termperature between about 0° C. and 100°C., most preferably between about 20° C. and 70° C.

The C-21 alcohol of formula IX is converted in the next step to a C-21halide or sulfonate ester of the formula ##STR13## wherein F, R, R¹, R²and X are as above;

The compound wherein X is p-toluenesulfonyloxy is especially preferred.

To prepare a compound wherein the C-21 substituent is a substitutedsulfonyloxy group, one would react the previously-mentioned C-21 alcoholof formula IX with a properly-substituted sulfonyl halide in thepresence of a base according to methods known in the art. Thepreparation of compounds wherein the C-21 substituent is iodo, bromo orchloro can be accomplished either by direct conversion of the C-21alcohol of formula IX to the desired halo group by means of ahalogenating agent such as, for example, phosphorus tribromide,according to methods well known in the art or by reaction of one of theC-21 sulfonate esters with a halide ion containing compound. Forexample, the C-21 sulfonate ester compound where the ester substituentis tosyloxy may be reacted with an alkali metal bromide or iodide, forexample, potassium bromide or potassium iodide, to afford the C-21halide compound where the halide is bromo or iodo, respectively. All ofthese interconversions to prepare the C-21 halide and C-21 sulfonateester compounds are standard in the art for the preparation of primaryalkyl halides and sulfonate esters from primary alcohols.

In the next step, the C-21 halide or sulfonate ester of formula X isconverted to a compound of the formula ##STR14## wherein F, R, R¹, andR² are as above; by reaction with a complex metal hydride reducingagent.

Suitable complex metal hydride reducing agents for this purpose includemetal aluminum hydrides such as lithium aluminum hydride; mono-, di- ortri(lower alkoxy) alkali metal aluminum hydride such as, for example,lithium tri(tert-butoxy) aluminum hydride; mono-, di- or tri(loweralkoxy lower alkoxy) aluminum metal hydrides such as, for example,sodium bis(2-methoxyethoxy) aluminum hydride; di(lower alkyl) aluminumhydrides such as, for example, diisobutyl aluminum hydride; and soforth. A particularly-preferred complex metal hydride reducing agent forthis purpose is lithium aluminum hydride. Suitable solvents for thereduction include ethers such as diethyl ether, tetrahydrofuran,dimethoxyethane and dioxane. The reduction is normally carried out at atemperature between about room temperature and about 100° C., mostpreferably between about 35° C. and 70° C. Other suitable reducingagents, particularly when the C-21 halide is iodo or bromo, are alkalimetal cyanoborohydrides such as, for example, sodium cyanoborohydride(sodium cyanotrihydroborate); tri(lower alkyl) tin hydrides such astri-n-butyltin hydride; and tri(aryl) tin hydrides such as triphenyltinhydride; and so forth. A particularly-preferred complex metal reducingagent is tri-n-butyltin hydride. Suitable solvents for the reductioninclude ethers such as diethyl ether, tetrahydrofuran, dimethoxyethaneand dioxane. The reduction is normally carried out at a temperaturebetween about -20° C. and 80° C., most preferably between about 0° C.and 40° C.

The substituted cholesterol derivatives of formula IV are deprotected byremoval of the protecting groups with a strong acid in a protic solventor with a tri-lower alkylsilyl iodide so as to yield[1α,3β-24,24-difluorocholest-5-en-1,3,25-triol of the formula ##STR15##wherein F is as above.

Suitable strong acids for this purpose include mineral acids such ashydrochloric or sulfuric acid; and organic sulfonic acids such asp-toluenesulfonic acid. Suitable protic solvents include alcohols suchas methanol and ethanol. It is preferable to carry out the removal ofthe aforementioned protecting groups at a temperature between about -10°C. and about 80° C., most preferably between about 0° C. and 40° C.Suitable tri-lower alkylsilyl iodides include trimethylsilyl iodide inan inert organic solvent such as hexane or methylene chloride, or anaromatic solvent such as benzene or toluene at -20° C. to the refluxtemperature of the solvent, preferably at 0° C.-50° C. under an inertatmosphere.

The [1α,3β]-24,24-difluorocholest-5-en-1,3,25-triol of formula IV' isthen alkanoylated to the compound of the formula ##STR16## wherein F,R', R^(1'), and R^(2') are as above, by methods well known in the art.For example, to selectively diacylate the 1α- and 3β-hydroxy groups ofcompound IV' to give IV-A, one may employ an acylating agent such asacetic anhydride, phenyl acetyl chloride or benzoyl chloride andtriethylamine or pyridine solvent at temperatures in the range of about-10° C. to about 50° C., most preferably at a temperature from 0° C. 3025° C.

The [1α,3β]-24,24,-difluorocholest-5-en-1,3,25-triol of formula IV' canalso be triacylated to a compound of the formula IV-A by employing theabove-mentioned acylating agents, solvents, and reaction temperaturesand employing a catalytic amount of 4-dimethylaminopyridine catalyst orby heating the above reaction mixtures to the 100°-150° with excessacylating agent.

The substituted cholesterol derivatives of formula IV-A are nextallylically halogenated to a mixture of 7α- and 7β-halocholesterols ofthe formula ##STR17## wherein F, R', R^(1'), R^(2') and X" are as above.

The halogenation reaction is accomplished using a suitable halogenationagent such as 1,3-dibromo-5,5-dimethylhydantoin, N-chlorosuccinimide,N-iodosuccinimide, N-bromoacetamide and the like dissolved in asaturated aliphatic hydrocarbon or halocarbon such as hexane or carbontetrachloride in the presence of an acid scavenger such as sodiumbicarbonate or sodium carbonate at the boiling point of the reactionmedium to give a mixture of 7α- and 7β-halocholesterols which is used inthe following dehydrohalogenation step without separation of the7α-halo-isomer from the 7β-isomer.

The 7α- and 7β-halocholesterol mixture of formula XI-A is converted tothe steroid 5,7-diene of the formula ##STR18## wherein F, R', R^(1'),and R^(2') are as above, by a dehydrohalogenation step. Thedehydrohalogenation of the crude mixture of 7α- and 7β-halocholesterolsis effected by heteroaromatic and aliphatic tertiary amines, pyridinesand alkylated pyridines such as picolines, lutidines and collidines;suitable aliphatic tertiary amines are triethylamine, tripropylamine,1,5-diazabicyclo (4.3.0) non-5-ene, 1,4-diazabicyclo(2.2.2) octane andthe like; s-collidine being preferred. Trialkylphosphites are alsouseful in the dehydrohalogenation step. Suitable inert organic solventsinclude aromatic and aliphatic organic solvents such as benzene,toluene, xylene, decalin and the like. Xylene is the preferred solvent.The reaction proceeds readily at temperatures from about 50° C. to thereflux temperature of the reaction medium, most readily at the refluxtemperature of the solvent system. The desired steroid 5,7-diene offormula XII-A can be isolated by the usual chemical and physical meanssuch as chromatography and in this manner can be separated from anyundesired impurities.

In the next step, the steroid 5,7-diene of formula XII-A is convertedinto the protected precholecalciferol compound of the formula ##STR19##wherein F,R',R^(1'), and R^(2') are as above, by irradiation under aninert atmosphere by means of a mercury lamp equipped with a glasscooling finger at a temperature range of about -40° C. to about 25° C.,about -20° C. to about 10° C. being the preferred irradiationtemperature range for the period of time necessary to effect about 50%conversion of the starting material. Suitable inert atmospheres includenitrogen, helium, argon and the like. Suitable source of irradiationenergy include high- and low-pressure mercury, xenon-mercury andthallium-mercury lamps. High-pressure mercury lamps are preferred. A 450W Hanovia high-pressure mercury lamp is the most preferred source ofirradiation energy. Suitable inert organic solvent systems for theirradiation include mixtures of saturated aliphatic hydrocarbons such aspentane, hexane, isooctane and the like and ethereal solvents such asdiethyl ether, dimethoxyethane, tetrahydrofuran, dioxane and the like.Preferred mixtures contain hexane and tetrahydrofuran.

Upon completion of the irradiation, the solvents are removed byevaporation, and the residue is separated into the pure protectedprecholecalciferol of formula XIII-A and pure unchanged steroid5,7-diene of formula XII-A on a high-performance liquid chromatographycolumn using a solid absorbent and an inert organic eluant. Suitableorganic eluants for the separation step include mixtures of hydrocarbonssuch as n-hexane, isooctane, cyclohexane and the like and esters such asethyl acetate, ethyl formate and the like. Suitable solid absorbentsinclude Porasil, Corasil, Biosil, Zorbax, Zorbax-Sil, Sil-X and thelike. A Waters Associates Chromatograph Model 202 using a four-foot by1-inch Porasil A column and a mixture of n-hexane:ethyl acetate as theeluant is the preferred high-performance liquid chromatographic system.Alternatively, the steroid 5,7-diene of formula XII-A is converted intothe protected pre-cholecalciferol of formula XIII-A by the processdisclosed in British Patent specification 1,592,170 published Jul. 1,1981.

The protected precholecalciferol of formula XIII-A is saponified to theprecholecalciferol of the formula ##STR20## wherein F is as above.

The saponification of the compound of formula XIII-A is conducted bytreatment with strong bases in protic solvents to yield the compound offormula XIV. Suitable bases include alkali and alkaline earthhydroxides, alkoxides such as methoxides, ethoxides and the like.Potassium hydroxide is most preferred. Suitable solvents includealcohols such as methanol and ethanol and water containing a misciblecosolvent to help solubilize the organic reactants, for example,, anether such as tetrahydrofuran or dimethoxyethane. Methanol is mostpreferred. It is preferable to carry out the removal of the protectinggroups R', R^(1') and R^(2') of formula XIII-A at a temperature betweenabout -20° C. and about 60° C., most preferably between about -5° C. and30° C. It is also preferable to perform the saponification under aninert atmosphere of nitrogen, argon and the like.

The last step in this sequence involves the thermal isomerization ofprecholecalciferol of formula XIV to give24,24-difluoro-1α,25-dihydroxycholecalciferol of the formula ##STR21##wherein F is as above, by heating at 50° C.-100° C. theprecholecalciferol of formula XIV in an inert solvent such as theethers, dioxane, tetrahydrofuran, dimethoxyethane and the like; thearomatic hydrocarbons such as benzene, toluene, and the like under aninert atmosphere such as argon, helium or nitrogen and the like bymethods well known in the art. See, for example, D. H. R. Barton et al.,J. Amer. Chem. Soc. 98(1973) 2748.

24,24-difluoro-1α,25 dihydroxycholecalciferol has shown activity in theanti-rachitogenic test in chicks, the diphosphonate procedure in rats,and in reduction of epiphyseal plate width in normal mice. The difluorocompound also has a long duration of activity in stimulation ofintestinal calcium absorption (96 hours after a single oral 100 nanogramdose). This long duration of activity makes 24,24-difluoro-1α,25dihydroxycholecalciferol useful for the prevention of milk fever incattle.

(a) Anti-Rachitogenic Activity in Chicks

One-day-old White Leghorn cockerels are placed on a vitamin D-deficientdiet which contains 1% calcium and 0.7% phosphorus and are housed underultraviolet-free lighting (General Electric F40G0 gold fluorescentlights). Compounds are dissolved in propylene glycol and administeredorally in a volume of 0.2 ml for 21 consecutive days to chicks which areone to two days of age at the start of treatment. Controls are treatedwith vehicle alone. Compounds are prepared in amber flasks and thesolutions are flushed with argon and refrigerated after each dosingperiod. Chicks are autopsied on the day after the last treatment day.Blood is collected for determination of serum calcium and phosphorus andtibia dry weight and ash weight are determined. Usually, ten chicks areused for each treatment group and for the control group. The results ofthe anti-rachitogenic activity assayare shown in Table I. The resultsshow that 24,24-difluoro-1α,25-dihydroxycholecalciferol possess potentanti-rachitogenic activity.

                  TABLE I                                                         ______________________________________                                        ANTI-RACHITOGENIC ACTIVITY OF 24,24-DIFLUORO-                                 1α.25-DIHYDROXY CHOLECALCIFEROL IN CHICKS                               MEAN TIBIA ASH WEIGHT (MG) ± S.E.                                          DOSE              24,24-difluoro-1α,25-dihydroxy-                       NG/CHICK/DAY      cholecalciferol                                             ______________________________________                                        0                 120.7 ± 5.9                                              1                 111.7 ± 5.8 NS                                           3                 151.5 ± 4.7 ***                                          10                227.1 ± 8.2 ***                                          30                244.8 ± 7.4 ***                                          21 DAYS ORAL DOSING                                                           9-10 CHICKS PER GROUP                                                         ______________________________________                                         *** STATISTICAL SIGNIFICANT RESULTS?                                          NS--NOT STATISTICALLY SIGNIFICANT?                                       

(b) Intestinal Calcium Absorption in Chicks

White Leghorn one-day-old cockerels are placed on the vitaminD-deficient diet and are housed under ultraviolet-free lighting for 21days. Chicks are then used to determine the effects of test compounds onintestinal calcium absorption. A single oral dose of test compounddissolved in propylene glycol is administered. At various times afterdosing, 2 uCi of ⁴⁵ Ca (chloride) is given orally, and serumradioactivity is measured 45 minutes after administration of theisotope. Ten chicks are used in each treatment and control group andvehicle-treated controls are included at each time period. The resultsof the intestinal calcium absorption assay are shown in Table II. Theresults show that 24,24-difluoro 1α,25-dihydroxycholecalciferol haspotent intestinal calcium absorption activity of long duration andconsequently possesses utility in the prevention of milk fever inpreparturient female ruminants.

                  TABLE II                                                        ______________________________________                                        COMPARISON OF 1α,25-DIHYDROXY CHOLECALCIFEROL                           AND 24,24-DIFLUORO-1α,25-DIHYDROXYCHOLECALCI-                           FEROL STIMULATION OF .sup.45 CA ABSORPTION                                    A - 1α,25-DIHYDROXYCHOLECALCIFEROL                                      B - 24,24-DIFLUORO-1α,25-DIHYDROXYCHOLECALCIFEROL                       TREATMENT     TIME    NO. OF     SERUM .sup.45 CA                             (ORAL)        (HR)    CHICKS     CPM/0.2 ML                                   ______________________________________                                        VEHICLE, 0.2 ML                                                                             24      11          992 ± 81                                 A - 0.1 MCG           11         1800 ± 181***                             B - 0.1 MCG           11         2064 ± 170***                             VEHICLE, 0.2 ML                                                                             48      11          769 ± 90                                 A - 0.1 MCG           11         1006 ± 133NS                              B - 0.1 MCG           11         1539 ± 99***                              VEHICLE, 0.2 ML                                                                             72      10          647 ± 69                                 A - 0.2 MCG           11          710 ± 62NS                               B - 0.1 MCG           11         1164 ± 90***                              VEHICLE, 0.2 ML                                                                             96      10          586 ± 70                                 B - 0.1 MCG           10          998 ± 61***                              VEHICLE, 0.2 ML                                                                             120     16          566 ± 39                                 B - 0.1 MCG           15          642 ± 38NS                               VEHICLE, 0.2 ML                                                                             144     16          696 ± 54                                 B - 0.1 MCG           16          672 ± 38NS                               ______________________________________                                         ***STATISTICALLY SIGNIFICANT RESULT                                           NS--NOT STATISTICALLY SIGNIFICANT                                        

(c) Prevention of EHDP-Induced Mineralization Block in Rats

Charles River CD male rats are treated for 10 consecutive days. Rats are21 days of age at the start of treatment. The disodium salt ofethane-1-hydroxy-1,1-diphosphonate (EHDP) is given subcutaneously oneach treatment day at a dose of 2 mg/0.2 ml/rat in distilled water. Testcompounds are administered orally on each treatment day in propyleneglycol (0.2 ml/rat). Rats are autopsied on the day after the lasttreatment day and tibias are processed by a modified von Kossa procedurebased upon silver impregnation of bone salts. Epiphyseal plate widthsare measured with a micrometer ocular using standard microscopicmagnification (35×). Activity is based upon dose-dependent narrowing ofthe widened epiphyseal plate induced by EHDP. Ten rats are used in eachtreatment group. Positive (EHDP alone) and negative (vehicles alone)control groups of ten rats each are included in each experiment. Theresults of the assay are shown in Table III. The results show that24,24-difluoro-1α,25-dihydroxycholecalciferol calcified the tibialepiphyseal plate in EHDP-blocked rats.

                  TABLE III                                                       ______________________________________                                        EFFECTS OF 24,24-DIFLUORO-1α,25-DIHYDROXY                               CHOLECALCIFEROL IN EHDP-TREATED RATS                                          MEAN TIBIA EPIPHYSEAL                                                         PLATE WIDTH (MICRA) ± S.E.                                                                     24,24-DIFLUORO-                                           DOSE                1α,25-DIHYDROXY-                                    NG/RAT/DAY          CHOLECALCIFEROL                                           ______________________________________                                        0                   1182 ± 51                                              1                   839 ± 18***                                            3                   674 ± 18***                                            10                  540 ± 16***                                            30                  412 ± 9***                                             VEHICLE CONTAINS (NO EHDP)                                                                        440 ± 2                                                10 DAYS ORAL DOSING                                                           10 RATS PER GROUP                                                             ______________________________________                                         ***STATISTICAL SIGNIFICANT RESULTS                                       

The compound of formula I can be administered for the treatment of milkfever in pregnant ruminant animals prior to parturation usingconventional formulations.

Sterile compositions for injection and/or topical administration can beformulated according to conventional practice by dissolving orsuspending the compound of formula I in a vehicle such as a 5-10%ethanol-water mixture, a naturally-occurring vegetable oil, such assesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or asynthetic fatty vehicle such as ethyl oleate or the like. For example, asuitable formulation for intravenous injection would be 2-3 ml of a5-10% ethanol-water solution containing 30-500 micrograms of a compoundof formula I. Such a formulation would preferably contain 30-400micrograms of a compound of formula I. Exemplary of a suitableformulation for topical administration would be a vegetable oil solutionor suspension containing 30-500 micrograms of a compound of formula I.Such a formulation would preferably contain 250-500 micrograms of acompound of formula I.

The compound of formula I can also be formulated for oral administrationby incorporation of 30-500 micrograms of a compound of formula I intofatty acid pellets.

The compound of formula I may also be formulated for intramuscularinjection by suspension of 50-500 micrograms of a compound of formula Iin a vehicle such as a 5-10% ethanol-water mixture or a 5-10% propyleneglycol-water mixture.

Buffers, preservatives, antioxidants and the like can be incorporatedinto the foregoing formulations as required.

The compound of formula I may be administered in dosages in the range ofabout 30-500 micrograms per day for the prevention of milk fever inpregnant ruminant animals. The compound of formula I is especiallyuseful for the prevention of milk fever in pregnant female bovines.

The Examples which follow further illustrate the disclosure. Alltemperatures are in degrees Centigrade unless otherwise stated.

EXAMPLE 1

To a solution of 10.8 ml of acetic anhydride and 10.8 mL of acetylchloride at 0° C. was added 10.80 g (0.070 mol) of 2,2-difluorosuccinicacid in several portions. The mixture was heated for 1 hr at 50° C. andcooled. The reaction was concentrated under reduced pressure to yield2,2-difluorosuccinic anhydride.

NMR (CDCl₃) δ 3.56 ppm (triplet, 2, J=15 Hz).

EXAMPLE 2

To 9.25 g (0.068 mol) of 2,2-difluorosuccinic anhydride at 0° C. wasadded dropwise 30 ml of dry absolute ethanol. The mixture was allowed towarm to 25° and was stirred for 18 hr. The mixture was evaporated underreduced pressure to yield ethyl 2,2-difluoro-3-carboxypropionate.

NMR (CDCl₃) δ 7.20 (broad, 1), 4.35 (quartet, 2, J=7 Hz), 3.31 (triplet,2, J=14 Hz), and 1.36 ppm (triplet, 3, J=7 Hz).

EXAMPLE 3

A solution of 123.0 mL (0.123 mol) of 1.0M of borane in tetrahydrofuranwas added dropwise at 0° to a mixture of 12.4 g (0.068 mol) of ethyl2,2-difluoro-3-carboxypropionate in 70 mL of dry tetrahydrofuran. Thereaction mixture was stirred at 0° for 18 hr then was quenched by adding40 mL of water dropwise. The solution was saturated with solid sodiumchloride and the product was isolated with ether. The ether layers werewashed with saturated aqueous sodium bicarbonate and brine. The solutionwas dried over anhydrous magnesium sulfate, filtered, and evaporated todryness to yield ethyl 2,2-difluoro-4-hydroxybutyrate.

NMR (CDCl₃) δ4.32 (quartet, 2, J=7 Hz), 3.87 (triplet, 2, J=6 Hz), 2.36(multiplet, 2) and 1.35 ppm (triplet, 3 J=6 Hz).

EXAMPLE 4

To a solution of 311 mL of 1.6M ethereal methyllithium (0.498 mol) of 0°was added dropwise 11.16 g (0.066 mol) of ethyl2,2-difluoro-4-hydroxybutanoate in 150 mL of ether. The mixture wasstirred at 0° C. for 1 hr and at 25° C. for 17 hr. The mixture wasquenched by adding 14 mL of saturated brine at 0° C. The mixture waspoured into saturated brine and the product was isolated with ether. Theether layers were dried over anhydrous magnesium sulfate, filtered, andevaporated to dryness. The residue was chromatographed on 0.06-0.20 mmof silica gel to give 4-methyl-3,3-difluoro-1,4-pentanediol.

NMR (CDCl₃) δ 3.84 (triplet, 2, J=6 Hz), 2.75 (broad, OH), 2.22(multiplet, 2), and 1.31 ppm (triplet, 6, J=1 Hz).

EXAMPLE 5

To a mixture of 6.92 g (0.045 mol) of4-methyl-3,3-difluoro-1,4-pentanediol and 28 mL of pyridine at 0° C. wasadded 16 mL of acetic anhydride and the mixture was stirred at 0° C. for1 hr and at 25° C. for 1 hr. The mixture was added to 15 mL of methanoland the solution was evaporated to dryness to yield oily4-methyl-3,3-difluoro-1,4-pentanediol 1-acetate.

NMR (CDCl₃) δ4.40 (triplet, 2, J=7 Hz), 2.30 (multiplet, 2), 2.06(singlet, 3) and 1.33 ppm (triplet, 6, J=1 Hz).

EXAMPLE 6

A mixture of 7.77 g (0.040 mol) of 4-methyl-3,3-difluoro-1,4-pentanediol1-acetate, 74 mL of ethyl vinyl ether and 0.50 g of p-toluenesulfonicacid monohydrate were stirred at -45° C. for 1 hr. The mixture wasquenched by adding 11 mL of triethylamine and evaporated to dryness. Theresidue was taken up in ether. This solution was successively washedwith saturated aqueous sodium bicarbonate solution and saturated brine.The ether phase was dried over anhydrous magnesium sulfate, filtered andevaporated to dryness to yield an oil containing4-(1-ethoxyethoxy)-3,3-difluoro-4-methyl-1-pentanol 1-acetate.

EXAMPLE 7

To a mixture of 2.25 g (0.059 mol) of lithium aluminum hydride in 50 mLof ether at 0° C. was added dropwise, 11.30 g (0.040 mol) of4-(1-ethoxyethoxy)-3, 3-difluoro-4-methyl-1-pentanol 1-acetate in 130 mLof ether. The mixture was heated at reflux (35° C.) for 3 hr and thenwas recooled to 0° C. The mixture was quenched by adding dropwise 4.5 mLof water followed by 3.6 mL of 10% aqueous sodium hydroxide. The mixturewas stirred at 25° C. for 0.5 hr and was filtered. Evaporation ofsolvent and column chromatography of the residue on 0.06-0.20 mm silicagel afforded 4-(1-ethoxyethoxy)-3,3-difluoro-4-methyl-1-pentanol.

NMR (CDCl₃) δ 5.00 (quartet, 1, J=7 Hz), 3.86 (broad, 2), 3.51 (quartet,2, J=7 Hz), 2.25 (multiplet, 2), 1.35 (triplet, 6, J=1 Hz), 1.30(doublet, 3, J=7 Hz), and 1.18 ppm (triplet, 3, J=7 Hz).

EXAMPLE 8

A mixture of 1.00 g (0.0044 mol) of4-(1-ethoxyethoxy)-3,3-difluoro-4-methyl-1-pentanol, 4 mL of pyridineand 1.27 g (0.0066 mol) of p-toluenesulfonyl chloride was stirred at 0°C. for 18 hr. The mixture was quenched with ice chips. The mixture wasthen poured into water and extracted with methylene chloride. Theorganic phase was sequentially washed with 10% aqueous sulfuric acid andsaturated aqueous sodium bicarbonate solution. The organic phase wasdried over anhydrous magnesium sulfate, filtered and evaporated todryness to yield oily4-(1-ethoxyethoxy)-3,3-difluoro-4-methyl-1-pentanol1-(4-methylbenzenesulfonate).

NMR (CDCl₃) δ 4.93 (quartet, 1, J=7 Hz), 4.27 (triplet, 2, J=7 Hz), 3.45(quartet, 2, J=7 Hz), 2.45 (singlet, 3), 2.36 (multiplet, 2), 1.27(triplet, 6, J=1 Hz), 1.24 (doublet, 3, J=7 Hz), and 1.15 ppm (triplet,3, J=7 Hz).

EXAMPLE 9

A mixture of 3.50 g (0.0087 mol) of4-(1-ethoxyethoxy)-3,3-difluoro-4-methyl-1-pentanol1-(4-methylbenzenesulfonate), 40 mL of acetone, 0.5 mL ofdiisopropylethylamine and 13.10 g (0.087 mol) of sodium iodide wasstirred at 25° C. for 64 hr. The mixture was evaporated to dryness. Theresidue was partitioned between 5% aqueous sodium sulfite solution andmethylene chloride. The organic phase was washed with saturated aqueoussodium bicarbonate solution. The organic phase was then dried overanhydrous magnesium sulfate, filtered, and evaporated to dryness. Theresidue was chromatographed on 0.06-0.20 mm silica gel to yield4-(1-ethoxyethoxy)-3,3-difluoro-1-iodo-4-methylpentane.

NMR (CDCl₃) δ 4.98 (quartet, 1, J=7 Hz), 3.50 (quartet, 2, J=7 Hz), 3.28(multiplet, 2), 2.60 (multiplet, 2), 1.32 (triplet, 6, J=1 Hz), 1.28(doublet, 2, J=7 Hz), and 1.18 ppm (triplet, 3, J=7 Hz).

EXAMPLE 10

A mixture of 0.91 g (0.0030 mol) of 1α,3β-dihydroxyandrost-5-en-17-one[R. M. Dodson, A. H. Goldkamp and R. D. Muir, J.Amer.Chem.Soc., 82, 4026(1960)], 15 mL of tetrahydrofuran, 1.26 g (0.015 mol) of3,4-dihydro-2H-pyran and 0.028 g of p-toluenesulfonic acid monohydratewas stirred at 25° for 18 hr. The mixture was diluted with methylenechloride. This solution was then washed with saturated aqueous sodiumbicarbonate solution. The organic phase was dried over anhydrousmagnesium sulfate, filtered and evaporated to dryness to yield oily[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]androst-5-en-17-one,[α]_(D) ²⁰ +34.3° (c 1, CHCl₃).

EXAMPLE 11

To a mixture of 1.00 g (0.0021 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]androst-5-en-17-one, 1.94g (0.0087 mol) of triethyl phosphonoacetate, and 14 ml of ethyl alcoholwas added 0.68 g (0.010 mol) of sodium ethoxide in 7 mL of ethanol. Themixture was stirred at reflux (80° C.) for 18 hr and cooled. The mixturewas concentrated under reduced pressure. The residue was partitionedbetween water and ether and the organic phase was washed with saturatedbrine. The organic phase was then dried over anhydrous magnesiumsulfate, filtered, and evaporated to dryness. The residue waschromatographed on 0.06-0.20 mm silica gel to yield[1α,3β,17(20)E]-1,3-bis[(tetra-2H-pyran-2-yl)oxy]pregna-5,17(20)-dien-21-oicacid ethyl ester [α]_(D) ²⁰ -8° (c 1, CHCl₃).

EXAMPLE 12

A mixture of 0.32 g (0.00059 mol) of[1α,3β,17(20E)]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]pregna-5,17(20)-dien-21-oicacid ethyl ester, 0.10 g of platinum oxide, and 20 mL of ethanol wasstirred in 1 atmosphere of hydrogen for 2 hr. The mixture was filteredthrough a pad of diatomaceous earth and the solids were washed withethanol. The combined filtrates were evaporated to dryness to yield oily[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]pregn-5-en-21-oic acidethyl ester, [α]_(n) ²³ -13° (c 1, CHCl₃).

EXAMPLE 13

To a solution of 1.0 mL of diisopropylamine in 3 mL of tetrahydrofuranat -30° C. was added 3.82 mL (0.0061 mol) of 1.6M of butyllithium inhexane. After stirring for 0.5 hr, 3.03 g (0.0056 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]pregn-5-en-21-oic acidethyl ether in 30 mL of tetrahydrofuran was added dropwise to yield thelithium enolate of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]pregn-5-en-21-oic acidethyl ester. The mixture was stirred for 1 hr at -30° C. and cooled to-70° C. A solution of 2.30 g (0.0068 mol) of4-(1-ethoxyethoxy)-3,3-difluoro-1-iodo-4-methylpentane in 5 mL ofhexamethylphosphoramie was added dropwise. The mixture was stirred at-70° C. for 1 hr and was allowed to warm to 25° C. and stir for 1 hr.The mixture was then diluted with 9:1 hexane-ether. The solution waswashed with water, and saturated brine. The organic phase was dried overanhydrous magnesium sulfate, filtered, and evaporated to dryness. Theresidue was purified by column chromatography on 0.06-0.20 mm silica gelto give[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-oicacid ethyl ester, [α]_(D) ²⁰ +10° (c 1, CHCl₃).

EXAMPLE 14

To a mixture of 0.20 g (0.0053 mol) of lithium aluminum hydride and 10mL of tetrahydrofuran at 0° C. was added 2.55 g (0.0034 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-oicacid ethyl ester in 35 mL of tetrahydrofuran. The mixture was heated at50° C. for 1.5 hr, recooled to 0° C., and diluted with 120 mL of ether.The mixture was then quenched with the dropwise addition of 0.40 ml ofwater and 0.32 mL of 10% aqueous sodium hydroxide. The mixture wasstirred at 25° C. for 1 hr and was filtered. The solids were trituratedwith ether and filtered. Evaporation of solvent afforded[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21ol,[α]_(D) ²⁰ +4° (c 1, CHCl₃).

EXAMPLE 15

A mixture of 2.37 g (0.0033 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-ol,7 mL of pyridine and 1.27 g (0.0067 mol) of p-toluenesulfonyl chloridewas stirred at 0° C. for 18 hr. The mixture was quenched with ice chips.The mixture was then poured into water and extracted with methylenechloride. The organic phase was washed with 10% aqueous sulfuric acidand saturated aqueous sodium bicarbonate solution. The organic layer wasdried over anhydrous magnesium sulfate, filtered and evaporated todryness to yield oily[1α,3β]-1,3-bis-[(tetrahydro-2H-pyran-2yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-ol21-(4-methylbenzenesulfonate), [α]_(D) ²⁰ +6° (c 1, CHCl₃).

EXAMPLE 16

A mixture of 0.221 g (0.00025 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-ol21-(4-methylbenzenesulfonate), and 0.150 g (0.0010 mol) of sodium iodidein 2 mL of acetone was heated at 50° for 18 hr and cooled. The mixturewas poured into water and the product was isolated with methylenechloride. The organic layers were washed with aqueous sodium sulfitesolution, and saturated aqueous sodium bicarbonate solution. The organiclayers were dried over anhydrous magnesium sulfate, filtered andevaporated to dryness to yield[1α,3β]-bis-[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluoro-21-iodocholest-5-ene.

EXAMPLE 17

A. A mixture of 0.410 g (0.0108 mol) of lithium aluminum hydride, 50 mLof tetrahydrofuran and 2.98 g (0.0033 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-en-21-ol21-(4-methylbenzenesulfonate) was heated at reflux (60°) for 1 hr andcooled to 0° C. The mixture was diluted with 120 mL of ether andquenched with the dropwise addition of 0.82 mL of water and 0.65 mL of10% aqueous sodium hydroxide solution. The mixture was then stirred for1 hr and filtered. The solids were triturated with ether and filtered.The combined filtrates were evaporated to dryness and chromatographed on0.06-0.20 mm silica gel to yield[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(2-ethoxyethoxy)-24,24-difluorocholest-5-ene,[α]_(D) ¹⁶ +2° (c 1, CHCl₃).

B. By an alternative procedure, a mixture of 0.205 g (0.00025 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluoro-21-iodocholest-5-ene,0.987 g (0.00030 mol) of tri-n-butyltin hydride and 3 mL oftetrahydrofuran were stirred at 25° C. for 18 hr under an argonatmosphere. The mixture was evaporated to dryness and the residue waspurified by chromatography on 0.06-0.20 mm silica gel to yield[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-ene,[α]_(D) ¹⁸ +2° (c 1, CHCl₃).

EXAMPLE 18

A mixture of 1.87 g (0.0027 mol) of[1α,3β]-1,3-bis[(tetrahydro-2H-pyran-2-yl)oxy]-25-(1-ethoxyethoxy)-24,24-difluorocholest-5-ene,70 mL of methanol and 0.30 g of p-toluenesulfonic acid monohydrate wasstirred at 25° C. for 4 hr. The mixture was quenched by adding 1.0 g ofsodium bicarbonate and stirring for 0.5 hr. The mixture was thenevaporated to dryness. The residue was triturated with ethyl acetate,filtered, and evaporated to dryness. The crude solid was recrystallizedfrom ethyl acetate to yield[1α,3β]-24,24-difluorocholest-5-en-1,3,25-triol, mp 177°-179°, [α]_(D)²¹ -15° (c 0.6, MeOH).

EXAMPLE 19

A mixture of 0.86 g (0.0019 mol) of[1α,3β]-24,24-difluorocholest-5-en-1,3,25-triol, 7 mL of pyridine and 4mL of acetic anhydride were stirred at 0° C. for 1 hr and at 25° C. for30 hr. The mixture was diluted with 20 mL of methanol at 0° C. andevaporated to dryness. The residue was then dissolved in methylenechloride. This solution was washed with 10% aqueous sulfuric acid andsaturated sodium bicarbonate solution. The organic phase was dried overanhydrous magnesium sulfate, filtered, and evaporated to dryness toyield [1α,3β]-24,24-difluorocholest-5-en-1,3,25-triol, 1,3-diacetate,[α]_(D) ²² -20° (c 1, CHCl₃).

EXAMPLE 20

A mixture of 1.05 g (0.0019 mol) of[1α,3β]-24,24-difluorocholest-5-en-1,3,25-triol 1,3-diacetate, 0.84 g ofsodium bicarbonate, 0.357 g (0.0012 mol) of1,3-dibromo-5,5-dimethylhydantoin and 40 mL of hexane was heated atreflux (80° C.) for 1 hr and cooled. The mixture was filtered and thesolids were triturated with hexane and filtered. The filtrates wereevaporated to dryness to yield[1α,3β,7ξ]-7-bromo-24,24-difluorocholest-5-en-1,3,25-triol1,3-diacetate.

EXAMPLE 21

A mixture of 1.33 g (0.0019 mol) of[1α,3β,7ξ]-7-bromo-24,24-difluorocholest-5-en-1,3,25-triol1,3-diacetate, 0.8 mL of s-collidine and 10 mL of xylene was heated atreflux (140° C.) for 0.5 hr and cooled. The mixture was diluted with 30mL of benzene. This solution was washed with 10% aqueous sulfuric acid,and saturated aqueous sodium bicarbonate solution. The organic phase wasdried over anhydrous magnesium sulfate, filtered, and evaporated todryness. The residue was purified by column chromatography on 0.06-0.20mm silica gel to yield[1α,3β]-24,24-difluorocholesta-5,7-dien-1,3,25-triol 1,3-diacetate,[α]_(D) ²⁵ -31° (c 0.4, CHCl₃), mp 130°-132°. UV max (C₂ H₅ OH) 261 (ε7750), 270 (ε 11,200), 281 (ε 12,100) and 293 (ε 7100).

EXAMPLE 22

A mixture of 0.210 g (0.0004 mol) of[1α,3β]-24,24-difluorocholesta-5,7-dien-1,3,25-triol 1,3-diacetate in 40mL of hexane and 10 mL of tetrahydrofuran was irradiated for 0.5 hrunder argon at -5° C. using a 450 W Hanovia high pressure mercury lamp,cooled with a Vycor-glass cooling finger. The solution was evaporated todryness and the residue was purified with a Waters Associates Liquidchromatograph Model 202 using 4'×1" silica gel column and a 3:1 mixtureof n-hexane-ethyl acetate as eluant to give[1α,3β,6Z]-24,24-difluoro-9,10-secocholest-5(10),6,8-trien-1,3,25-triol1,3-diacetate. UV max (C₂ H₅ OH) 260 nm (ε 10,700).

EXAMPLE 23

A solution of 0.071 g (0.00013 mol) of[1α,3β,6Z]-24,24-difluoro-9,10-secocholesta-5(10),6,8-trien-1,3,25-triol1,3-diacetate, and 7 mL of 1.5% potassium hydroxide in methanol wasstirred at 0° C. for 4 hr. The mixture was neutralized to pH 7.5 withglacial acetic acid in methanol. The solution was then evaporated todryness at 0° C. The residue was partitioned between ethyl acetate andwater. The organic phase was washed with saturated brine and dried overanhydrous sodium sulfate. The mixture was filtered and evaporated todryness to yield[1α,3β,6Z]-24,24-difluoro-9,10-secocholesta-5(10),6,3-trien-1,3,25-triol.UV max (C₂ H₅ OH) 260 nm (ε 10,800). The reaction was carried out underan argon atmosphere.

EXAMPLE 24

A mixture of 0.063 g (0.00013 mol) of[1α,3β,6Z]-24,24-difluoro-9,10-secocholesta-5-(10),6,8-trien-1,3,25-triol,and 10 mL of p-dioxane was heated at reflux (100° C.) for 1 hr andcooled. The mixture was then evaporated to dryness. The residue waspurified with a Waters Associates liquid chromatograph Model 202 using a4'×1" silica gel column and 3:1 ethyl acetate-hexane as eluant to give[1α,3β,5Z,7E]-24,24-difluoro-9,10-secocholesta-5,7,10(19)-trien-1,3,25-triol also known as24,24-difluoro-1α,25-dihydroxycholecalciferol, [α]_(D) ²¹ +49° (c 0.5,MeOH). UV max (C₂ H₅ OH) 265 nm (ε 15,500).

What we claim:
 1. A method for the prevention of milk fever in ruminantanimals which comprises administering to a pregnant ruminant animal aneffective amount of 24,24-difluoro-1α,25-dihydroxycholecalciferol priorto parturation.
 2. The method according to claim 1 wherein said ruminantanimal is a female bovine.
 3. The method according to claim 2 whereinsaid effective amount of 24,24-difluoro-1α,25-dihydroxycholecalciferolis administered about one to four days prior to parturation.
 4. Themethod according to claim 3 wherein said effective amount of24,24-difluoro-1α,25-dihydroxycholecalciferol is in the range of about30-500 micrograms.
 5. The method for treatment and prophylaxsis for milkfever disease in diary cattle which comprises administering to saidcattle prior to parturition a vitamin D derivative, which is24,24-difluoro1α,25-dihydroxy-vitamin D₃.
 6. The method of claim 5 inwhich the 24,24-difluoro-1α,25-dihydroxyvitamin D₃ is administered in anamount from about 0.1 to about 10 mg per animal from about 4 to 8 daysprior to calving.
 7. The method of claim 6 wherein the24,24-difluoro-1α,25-dihydroxyvitamin D₃ is administered by injection.8. The method of claim 7 wherein the injection is intramuscular.
 9. Themethod of claim 7 wherein the injection is subcutaneous.
 10. The methodof claim 6 wherein the 24,24-difluoro-1α,25-dihydroxyvitamin D₃ isadministered orally.